Targeted identification of immunogenic peptides

ABSTRACT

This invention relates generally to identifying peptide sequences involved in antibody binding to any protein for synthesis of vaccine treatments. This novel method allows for a more manageable vaccine peptide discovery and specific generation of unique immunogenic peptides from self-tumor associated proteins and/or foreign proteins from infectious organisms for specific and/or enhanced expression only in the presence of the antibody.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/714,865 entitled “Targeted Identification of Immunogenic Peptides”filed Sep. 8, 2005 and is a continuation of International ApplicationNo. PCT/US06/35171 filed Sep. 8, 2006, the entirety of each is herebyincorporated by reference.

RIGHTS IN THE INVENTION

This invention was made, in part, with support for the United Statesgovernment and the United States government may have an interest in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention, in the field of immunology/immunotherapy, vaccinediscovery and development, relates generally to the identification ofimmunogenic peptides from regions of proteins and molecules that areinvolved in the binding interactions with polyclonal and monoclonalantibodies and other specific binding peptides/molecules. The presentinvention is directed to methods for identification and use of thepeptides for preventing, suppressing and treating immune-relateddiseases. Specifically, the invention provides therapy that result inclinical improvement in cancer patients.

2. Description of the Background

Autoimmune diseases are characterized by an unwanted and unwarrantedattack by the immune system on the tissues of the host. While themechanism for progress of these diseases is not well understood, atleast some of the details with respect to antigen presentation areknown. It is thought that antigens, including autoantigens, areprocessed by antigen-presenting cells (APC), and the resulting fragmentsare then associated with one of the cell surface proteins encoded by themajor histocompatibility complex (MHC). As a result, recognition of apeptide antigen is said to be MHC “restricted.” When the MHC/antigenfragment complex binds to a complementary T cell receptor (TCR) on thesurface of a T lymphocyte, activation and proliferation of the clone orsubpopulation of T cells result bearing that particular TCR. Onceactivated, T cells have the capacity to regulate other cells of theimmune system which display the processed antigen and to destroy thecells or tissues which carry epitopes of the recognized antigen.

Antibody therapies in which antibodies are directed to MHC molecules andCD4 molecules have been generally successful in several animal models ofautoimmunity. However, these approaches may be too nonspecific andpotentially overly suppressive. This may be because 70% of T cells bearthe CD4 marker and because all T cell-mediated responses and mostantibody responses require MHC-associated antigen presentation.

A major difficulty with present approaches is that they require the useof complex biological preparations which do not comprise well-definedtherapeutic agents. Such preparations suffer from complex production andmaintenance requirements (e.g., the need for sterility and largequantities of medium for producing large number of “vaccine” T cells),and lack reproducibility from batch to batch. To be useful in humans, Tcell “vaccine” preparations must be both autologous and individuallyspecific. This means they must be uniquely tailored for each patient.Furthermore, the presence of additional antigens on the surface of suchT cells may result in a broader, possibly detrimental, immune responsenot limited to the desired T cell clones (Offner et al., J.Neuroimmunol. 21:13-22 (1989).

There is a need, therefore, for agents and pharmaceutical compositionswhich have the properties of specificity for the targeted immuneresponse. These agents and compositions should also have predictabilityin their selection, convenience and reproducibility of preparation, andsufficient definition in order to permit precise control of dosage.

An effective vaccine is capable of generating a long-lasting immunitywhile being relatively harmless to the recipient. Attenuated organismsand purified antigens from organisms have traditionally been used asvaccines. However, such agents often produce deleterious side effects orfail to protect against subsequent challenges. Because of the inherentdifficulties in growing pathogenic organisms and producing effectivevaccines from them, many viral, bacterial and parasitic diseases have noeffective vaccine.

A further difficulty with the use of peptides as vaccines is that, inmost instances, peptides alone are not good immunogens. It is a wellknown phenomenon that most immune responses to peptide antigens are Tcell-dependent. Accordingly, “carrier” molecules have been attached topeptide antigens that bind, for example, to B cell surfaceimmunoglobulin in order to generate a high affinity, IgG response. Inother words, nonresponsiveness to peptide antigens may sometimes beovercome by attaching another peptide that induces helper T cellactivity.

In general, peptides that induce helper T cell activity are generated byB cells from enzymatic digestion of native proteins internalized by wayof an antibody receptor. These T cell stimulating peptides are thenpresented on the surface of the B cell in association with class IImajor histocompatibility complex (MHC) molecules. In a similar fashion,peptides that induce cytotoxic T cell activity may be generated byaccessory cells, including B cells. These peptides are presented on thecell surface of accessory cells in association with class I MHCmolecules. As used herein, the term “T cell stimulatory peptide” meansany peptide which activates or stimulates T cells, including (but notlimited to) helper T cells and/or cytotoxic T cells.

Peptides represent a promising approach to the production and design ofvaccines. However, the difficulties in making peptides that induce thedesired immune response have hampered their success. This includes thedifficulties inherent in making peptides that closely mimic the nativestructure of antigenic determinants.

These antigenic determinants, or epitopes, of a protein antigenrepresent the sites that are recognized as binding sites by certainimmune components such as antibodies or immunocompetent cells. Whileepitopes are defined only in a functional sense, i.e. by their abilityto bind to antibodies or immunocompetent cells, there is a structuralbasis for their immunological activity.

Epitopes are classified as either being continuous and discontinuous.Discontinuous epitopes are composed of sequences of amino acidsthroughout an antigen and rely on the tertiary structure or folding ofthe protein to bring the sequences together and form the epitope. Incontrast, continuous epitopes are linear peptide fragments of theantigen that are able to bind to antibodies raised against the intactantigen.

Many antigens have been studied as possible serum markers for differenttypes of cancer because the serum concentration of the specific antigenmay be an indication of the cancer stage in an untreated person. Assuch, it would be advantageous to develop immunological reagents thatreact with the antigen. More specifically, it would be advantageous todevelop immunological reagents that react with the epitopes of theprotein antigen.

Conventional methods using biochemical and biophysical properties haveattempted to determine the location of probable peptide epitopes. Thesemethods include a careful screening of a protein's primary structure,searching for critical turns, helices, and even the folding of theprotein in the tertiary structure. Continuous epitopes are structurallyless complicated and therefore may be easier to locate. However, theability to predict the location, length and potency of the site islimited.

Various other methods have been used to identify and predict thelocation of continuous epitopes in proteins by analyzing certainfeatures of their primary structure. For example, parameters such ashydrophilicity, accessibility and mobility of short segments ofpolypeptide chains have been correlated with the location of epitopes.

Hydrophilicity has been used as the basis for determining proteinepitopes by analyzing an amino acid sequence in order to find the pointof greatest local hydrophilicity. As discussed in U.S. Pat. No.4,554,101, each amino acid is assigned a relative hydrophilicitynumerical value which is then averaged according to local hydrophilicityso that the locations of the highest local average hydrophilicity valuesrepresent the locations of the continuous epitopes. However, this methoddoes not provide any information as to the optimal length of thecontinuous epitope. Similarly, U.S. Pat. No. 6,780,598 B1 determines theimmunopotency of an epitope by providing a ranking system delineatingbetween dominant and subdominant epitopes.

Computer-driven algorithms have been devised to take advantage of thebiochemical properties of amino acids in a protein sequence by sortinginformation to search for T cell epitopes. These algorithms have beenused to search the amino acid sequence of a given protein forcharacteristics known to be common to immunogenic peptides. They canoften locate regions that are likely to induce cellular immune responsein vitro. Computer-driven algorithms can identify regions of proteinsthat contain epitopes which are less variable among geographic isolates,or regions of each geographic isolate's more variable proteins, orperform as a preliminary tool to evaluate the evolution of immuneresponse to an individual's own quasi species.

Peptides presented in conjunction with class I MHC molecules are derivedfrom foreign or self protein antigens that have been synthesized in thecytoplasm. Peptides presented with class II MHC molecules are usuallyderived from exogenous protein antigens. Peptides binding to class Imolecules are usually shorter (about 8-10 amino acid residues) thanthose that bind to class II molecules (8 to greater than 20 residues).

Identification of T cell epitopes within protein antigens hastraditionally been accomplished using a variety of methods. Theseinclude the use of whole and fragmented native or recombinant antigenicprotein, as well as the more commonly employed “overlapping peptide”method for the identification of T cell epitopes within protein antigenswhich involves the synthesis of overlapping peptides spanning the entiresequence of a given protein. Peptides are then tested for their capacityto stimulate T cell cytotoxic or proliferation responses in vitro.

The overlapping peptide method is both cost and labor intensive. Forexample, to perform an assay using 15 amino acid long peptidesoverlapping by 5 amino acids spanning a given antigen of length n (asmall subset of the possible 15-mers spanning the protein), one wouldneed to construct and assay (n/5)-1 peptides. For most types ofanalyses, this number would be prohibitive.

Accordingly, a simple method to identify immunogenic peptides fromregions of self-proteins and other proteins and molecules involved inbinding interactions with polyclonal and monoclonal antibodies isneeded.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts one embodiment of the invention in which a uniqueimmunogenic region of the HER-2/neu is identified.

FIG. 2 shows the Her2/neu sequence (SEQ ID NO: 1) and highlights theantibody-binding site of Herceptin to the Her2/new protein. Also shownis the E75 vaccine sequence (SEQ ID NO: 2) and the Her2 loops binding toHerceptin (SEQ ID NO: 3).

FIG. 3 shows HLA peptide motif search results and scoring results forSEQ ID NOs: 4-11.

FIG. 4 shows amino acid substitutions for increased binding affinity ofnative peptide vaccine development. A table indicating HLA peptide motifsearch results, as well as the scoring results for SEQ ID NOs: 2, 4, and12-18 are included.

FIG. 5 depicts a T2 Peptide-binding assay, in which 25 μg of peptidewere incubated with T2 cells, levels of HLA-A2 levels were measured byflow cytometry and are indicated as Mean Fluorescence Intensity units.

FIG. 6 is a table depicting an experiment looking at whether 2G-577- andE75-stimulated cells can lyse tumor cells, in prostate cancer, ovariancancer, and breast cancer, with results expressed as % lysis.

FIG. 7 is a table showing enhanced lysis of Herceptin-treated tumortargets by E75, GP2, 2G-577, 2V-577, and 2L-577-stimulated cells, withresults expressed as % lysis. These data suggest the benefit ofcombination therapy with Herceptin treatment and vaccine-induced T celltargeting for synergistic tumor cell killing.

FIG. 8 is a table showing enhanced lysis of Herceptin-treated tumortargets by 2G-577-stimulated cells from breast cancer patients, withresults expressed as % lysis.

SUMMARY OF THE INVENTION

The invention overcomes the problems and disadvantages associated withcurrent methods and provides tools and methods of generating an immuneresponse in a patient in need thereof.

One embodiment of the invention is directed to a method for synthesizingan immunogenic peptide from a self-protein comprising the steps ofidentifying one or more peptide sequences of a self protein that aredirectly or indirectly involved with antibody-binding, subjecting theone or more peptide sequences to an algorithm that identifies sequencessuspected of being immunogenic, screening all peptide fragments from theone or more peptide sequences, and identifying an immunogenic peptide ofthe protein fragment wherein the antibody-binding interaction ispolyclonal or monoclonal. Further, a patient is treated with theimmunogenic peptide to generate an immune response.

Another embodiment of the invention is directed to immunogenic peptidesidentified by the method described above.

Another embodiment of the invention is directed to an immunogenicpeptide that produces an immune response to a self-protein.

Another embodiment is directed to a method of presenting epitopes forrecognition by the immune system to generate an immune responsecomprising the steps of identifying a protein fragment that isrecognized by a pool of unused and immunoreactive T cells, subjectingthe protein fragment to an algorithm, identifying one or more specificsequences of the protein fragment that is immunogenic, synthesizing atleast one immunogenic peptide corresponding to the sequence and treatinga patient with the immunogenic peptide to generate an immune response.Further, an antibody that binds to the protein is generated.

Another embodiment of the invention is directed to immunogenic peptidesidentified by the method described above.

Another embodiment of the invention is directed to a vaccine comprisingthe immunogenic peptides described above.

Another embodiment of the invention is directed to a vaccine comprisingantibodies that react with the immunogenic peptides described above.

Another embodiment of the invention is directed to a method ofidentifying a vaccine treatment comprising the steps of binding anantibody to a protein molecule forming a complex, subjecting the complexto proteasome digestion, obtaining digestion products comprisingpeptides, and identifying an immunogenic peptide sequence from thedigestion products.

Another embodiment of the invention is directed to a method ofidentifying a patient-specific treatment comprising the steps ofobtaining a pre-existing immuno-reactive precursor to said patient'sAGIE/ABIE, culturing tumor cells obtained from said patient, incubatingthe cultured tumor cells that are reactive against generated antibodies,examining dataset responses in presence and absence of the generatedantibodies and identifying the patient-specific immunogenic epitopes.This method may include the generation of antibodies that are reactiveagainst self antigens, the generation of antibodies that are reactiveagainst foreign antigens, and/or the generation of antibodies, onceadministered to said patient, that are therapeutic or prophylactic.

Another embodiment of the invention is directed to a method ofidentifying a vaccine treatment comprising the steps of binding anantibody to a protein molecule with a specific binding activity forminga complex, subjecting the complex to proteasome digestion, obtainingdigestion producing comprising peptides, identifying an immunogenicpeptide sequence from the digestion products.

Other embodiments and technical advantages of the invention are setforth below and may be apparent from the drawings and the description ofthe invention which follow, or may be learned from the practice of theinvention.

DESCRIPTION OF THE INVENTION AND EXAMPLES

Treatments for complex diseases involving the immune system and immuneresponses caused by endogenous self antigens and/or foreign antigensthat are involved in producing autoimmune antibody are extremelydifficult to discover. Antigens involved with such diseases are eitherforeign or self antigens (or both). Administration of foreign antigensfor passive immunization can result in serum sickness-like immunecomplex diseases. Also, reactive T cells capable of recognizing selfpeptides are typically deleted or processed and destroyed. Thesepeptides, generated and displayed under normal and constitutiveconditions are degraded by cell protein degradation machinery resultingin the absence of an immune response.

A simple method for identification of immunogenic regions ofself-proteins and other proteins and molecules involved in the bindinginteractions with polyclonal and monoclonal antibodies has beensurprisingly discovered. From this method, new and unique epitopes aregenerated that are presented in the presence of bound ligands, such as,preferably antibodies. Once these immunogenic regions are identified,vaccines comprising the antigen, modifications of the antigen, orantibodies specifically reactive to the antigen or the modified antigencan be prepared. Thus, the present invention makes vaccine peptidediscovery manageable, and allows for the specific generation of uniqueimmunogenic peptides from self-tumor associated proteins that can beinduced or generated for specific expression in the presence of theantibody, allowing for vaccine and/or novel combination therapy.

The invention is described more fully herein and refers to manypreferred embodiments. This invention, however, should not be construedas limited to those embodiments.

The proteasome is a multi-subunit complex with proteolytic cleavageactivities that result in the generation of a wide variety of peptidesfrom proteins. The susceptibility of a given protein to the proteolyticactivities of the proteasome is dependent upon the various primary,secondary and tertiary structural and post-translational modificationsthat take place within the proteasome. These activities expose certainsequences or regions of the protein and not others, to the system.

In one embodiment of the invention, cancer cells are induced to expressimmunogenic peptides from unique or self tumor-specific antigens tostimulate anti-tumor immune responses for immunotherapy. Normally thesepeptides are not generated from self-proteins. Binding of antibodies (orother molecules) to the sites normally accessible and processed byproteasomes alters the pattern of accessibility and the resultingproteolytic cleavage pattern by the proteasome. Such alteration of thesite results in generation of novel peptides that may be intrinsicallyimmunogenic because they have not been previously expressed anddisplayed for the deletion of immuno-reactive T cells.

In one preferred embodiment, a unique immunogenic region of theHER-2/neu was identified. HER-2/neu is an over-expressed oncogenicprotein. Conventional vaccine strategies, normally effective inimmunizing patients, did not work with a “self-protein” such asHER-2/neu. Tolerance to self-proteins may only be directed to dominantepitopes of the protein and not the entire protein. Therefore,immunization to just a specific protein fragment, and not the entireprotein, alleviates this problem. This specific protein fragment islocated with in the sequence directly involved with antibody-bindinginteraction or in the proximity of that region.

After identifying this restricted, shorter peptide sequence, thesequence is subjected to algorithms to identify likely functionallyactive or target sequences or regions. Running algorithms on thissegment, as opposed to the whole segment, provides a manageable set ofpeptides to test as candidates for vaccine development. In the past,computer-driven tests were run on the entire sequence consuming muchtime, money and effort. The algorithms searched the amino acid sequenceprovided for characteristic immune response in vitro. Regions of theproteins identified as containing epitopes may be useful as a vaccine.

Next, treatment of the tumor cells with the antibody against theidentified peptide sequence was performed. Inducibility of the alteredturnover and subsequent generation of the newly identified peptides onlyin the tumor cells and only in the presence of the antibody gave it aspecific targeting and triggering feature that was controllable. SeeFIG. 1. An antibody booster can be given to increase thepeptide-specific cytotoxic T cell response. In cases where the antibodyalready existed, discovery of the peptide only was sufficient fortriggering the specific immune response.

The method to generate new and unique epitopes included theidentification of immunogenic peptides from regions of proteins andmolecules involved in the binding interactions with most any ligandincluding, but not limited to polyclonal and monoclonal antibodies,receptors, ligands and any molecule with high affinity to the antigen.These new epitopes were presented to antigen-processing cells in thepresence of bound ligand. In certain cases, binding protected theepitope or a portion of the epitope revealing the immunogenic portion ofthe antigen. These immunogenic regions become new and uniquely enhancedtargets for recognition by the immune system and for use in modulationof the immune responses for the treatment of disease states andimmunotherapy either by themselves, an antibody vaccines, or when usedin combination with other molecules or antibodies. Theseantibody-generated immunogenic epitopes (AGIE)/antibody-boundimmunogenic epitopes (ABIE) were useful for stimulating immune responsein cancer and infectious diseases. They can also suppress immuneresponses in autoimmune diseases and transplantation based upon theactivity of the antibody in modulating the immune response in thedirection of upregulation or down regulation.

Peptides varying in length including single chain antigen-bindingpolypeptides that can specifically interact, protect and/or modulate anygiven epitope such that it results in specifically protecting and/orenhancing the preservation and presentation of the epitope whensubjected to the various proteolytic activities of the antigenprocessing machinery of the proteasome and immunoproteasome subunits andcomplexes.

Another embodiment of this invention comprises a combination of specificantibodies and/or treatment with other molecules that involve the sameregions of the peptide sequences and targets unique populations ofcells. This technology allows for the development of novel vaccines madeof antibodies and/or antigen-binding fragments Fab or F(ab)′2-fragmentsbound to specific length peptides that contain immunogenic epitopes(s)of interest that are specifically processed and presented.

Specific epitopes of proteins may be protected, both intracellular andextracellular, that are normally destroyed by antigen processingmachinery of proteasomes and immunoproteasomes by single chainantibodies and/or peptides that can specifically bind to selected sitesequences so that these epitopes become novel targets that are developedas unique vaccines for the specific recognition of cancer cells. HSPsthat are released during inflammatory processes perform similaractivities and enhance immune responses because they were made to bindto many different peptide sequences non-covalently.

Another preferred embodiment of the invention is directed to a methodfor the identification of highly immunogenic peptides. These peptidesare used to enhance or suppress immune responses from normal cellproteins that are not processed and presented naturally because of theconstitutive destruction of these epitopes by the normal activities ofthe proteasome and immunoproteasome complexes. These peptides are usedfor generation for specific antibodies that bind and generate the novelpresentation of these epitopes for recognition by the immune system.

In a preferred example, a large pool of immunoreactive T cells that arepresent or may be generated that are capable of reacting with theseinert/naturally non-existent epitopes, but are not utilized and areconsidered “wasteful” because these epitopes are not being generatednormally. This allows access to the pools of unused T cells forspecifically directed therapeutic benefits. Again, computer-drivenalgorithms are run. Upon identification of the peptide sequencesinvolved with antibody-binding interaction, these peptides are used toimmunize animals and/or patients to generate specific antibodies thatbind the protein and generate these novel peptides for subsequent immunerecognition and response.

Another embodiment of the invention is directed to a direct method ofbinding antibody or antibodies to the protein molecules and/orpolypeptide regions and subjecting these bound and unbound/nativecomplexes to ex vivo or in vitro to all forms of the proteasomemachinery. Proteolytic digestion or cleavage products are obtained toobserve differential yield of peptides for use in vaccine development.However, there is a drawback to this method in that it is not fullyrepresentative of all “mechanisms and proteolytic activities” that mayoccur in vivo within the cell itself, thereby limiting application. Suchan approach however, is useful for the proof of concept in systems whereit provides clean reproducible and predictable results, e.g. usingvarious individual components of proteasomes and combinations thereofand then observing peptide patterns by mass spectroscopy analysis. It isalso useful for the definition of peptide patterns generated in thepresence and/or absence of proteasome inhibitors with and without boundantibody or antibodies.

Another embodiment of the present invention involves application of thisapproach for identification of patient-specific treatment. Optimalantibody choices are made for the patient based upon patient's personalpre-existing immuno-reactive precursors to the AGIE/ABIE. First, PBMC(peripheral blood mononuclear cells) are obtained from patient andculture in presence or absence of cytokines (eg IL-2, IL-7, and IL-15)in the presence of FCS (fetal calf serum) or autologous serum. Afteroptimal culture and expansion during a set number of days, the cells aretested in cytotoxicity assays against tumor cell lines that have beenpre-incubated in the presence and absence of specific antibodies orantibody combination(s). Datasets from responses seen specifically topresence of antibodies and those where individual antibodies do notyield a response, but the combinations that do are noted. Theepitopes/vaccine peptides predicted or defined by the binding-sites ofthe antibodies are tested for fine reactivities in determination ofwhich vaccines to use on patients.

Another embodiment of the invention is directed to identifying antigensthat are not expressed on the cell surface or for parts of protein thatare always cytoplasmic or for proteins that are completelyintracellular/intranuclear or cytoplasmic (e.g. p53, telomerase, etc.).Methods for expressing the “modulating” antibody or antibodies asendogenous proteins, so that they are able to bind these proteinsintracellularly and modulate their processing and presentation fromwithin. The endogenous expression methods included recombinant DNAexpression systems as well as viral/bacterial vector delivery andexpression systems.

Another embodiment of the invention is directed to “shuttling” in theantibodies or single-chain antigen-binding fragment encapsulated withinvarious delivery systems to include liposomes, micelles, nanoparticlesand others.

Generation of “protective” or “suppressive” antibody preparations orcombination capable of being administered passively for treatment ofdisease similar to the gamma-globulin shots were generated as polyclonalpreps or as specific combinations of selected antibodies against one ormore tumor or tissue specific antigens for that particular disease orcondition.

The present invention is not restricted to HLA-A2 or class I peptidesbecause one or more longer-length peptides from a known region orsequence can be used for the generation of AGIE/ABIE by the antibody orantibodies in question. The benefit is not having to be restricted byonly specific or limited HLA types (Class I and II) in terms of patientsthat can be treated. Furthermore, although the majority of Class I andClass II peptides are derived from the processing of endogenous andexogenous proteins respectively, the well described and acceptedmechanisms of “cross-presentation” allows for the presentation of allsources or peptides on both classes of MHC molecules.

In another embodiment of the invention, the invention is used foridentifying inducible vaccine responses by “discovering” the MAb/s thatwill generate AGIE/ABIE. This is achieved by first screening 10-mer or20-mer consecutive or overlapping peptides from antigen for the highestprecursor T-cell responses present in cancer and/or normal individuals.These “ultra-immunogenic” peptides are then used for generation of MAbin mice. These MAbs are then tested for ability to generate AGIE/ABIEspecific responses in MAb-treated targets. MAbs that are promising arethen humanized for therapeutic purposes. To ensure involvement of T cellspecific immune responses the promising Fab′ fragments are first used toeliminate purely antibody-dependent cell-mediated cytotoxicity(ADCC)-mediated activity.

Another way of specifically identifying peptides capable of generatingantibodies that induce AGIE/ABIE that are most protective is by themethod of screening serum sample from high-risk cancer individuals(those with family history of cancer, e.g. smokers who do and do not getlung cancer, patients who are ‘cured’) who do and do not develop cancer(or from progressor vs non-progressors in the case of AIDS) to look forAb responses against the overlapping peptides from specific wellcharacterized or defined tumor antigens (HER2/neu, prostate specificantigen or PSA, prostate specific membrane antigen or PSMA, Tyrosinase,melanoma Ags, etc.) that are present in the protected individuals orpresent in fully recovered/cured cancer patients. Based on the specificAb responses that are found to be uniquely present or predominantlypresent in the ‘protected’ individuals, peptides are targeted forgeneration or ‘discovering’ of MAb that generated AGIE/ABIE. Alldescriptions herein were done for both Class I and Class II epitopes andresponses.

In a preferred embodiment, data of appropriate combinations ofantibodies from published sources with well documented ‘pre-existing’corresponding CD4-helper and CD8 CTL-specific responses is used fordeveloping a vaccine for the AIDS virus and other immunotherapytreatment using the approach from the present invention. The HIVMolecular Immunology Database provided by the National Institute ofAllergy and Infectious Diseases at www.hiv.lanl.gov offers a currentcomprehensive listing of defined HIV epitopes. The website also includesthe exact epitope and binding site of all known monoclonal andpolyclonal antibodies to the protein sequences as well as neutralizationactivity.

The present invention provides enhanced lytic activity ofantibody-treated tumor cells by vaccines identified from the bindingsites of these antibodies. The present invention also provides a novelindication for antibodies already in clinical use for cancer treatmentas combination therapy furthering development of vaccine treatmentdiscovery.

EXAMPLE

The binding of trastuzumab (Herceptin) to HER2/neu for the developmentof novel vaccines against HER2/neu-expressing tumors was studied. TheAb-binding site of trastuzumab (Tz) was analyzed for peptides that bindHLA-A2 and A3. A peptide Her577 (aa: 577-585) within the Ab-binding siteon HER2/neu was identified, synthesized and tested. T2 HLA-stabilizationassays were performed to confirm HLA-A2 binding activity of Her577 byflow cytometry. PBMC from 9 healthy donors were stimulated with Her577and tested in ⁵¹Cr-release cytotoxicity (CTX) assays against 3HER2/neu-expressing tumor cell lines. Her577-stimulated PBMC from 5HLA-A2⁺ healthy donors were also tested in CTX with HER2/neu⁺ targetspre-treated with Tz. Simultaneous experiments were done with E75 andGP2, two other immunogenic peptides from HER2/neu.

The Her577 peptide bound HLA-A2 comparable to E75 and GP2 with meanfluorescence intensity values of 1275, 1151 and 682, respectively. Theaverage specific CTX by Her577-stimulated cytotoxic T lymphocytes (CTL)against LNCaP, SKOV-3 and MCF-7 tumor cells was found to be similar toCTX achieved with E75- and GP2-stimulated CTL (% lysis; Table 1).

TABLE 1 LNCaP SKOV-3 MCF-7 (HLA-A2⁺) (HLA-A3⁺) (HLA-A2⁺) (n = 2) (n = 2)(n = 5) Her577 30 ± 3 49 ± 28 45 ± 3 E75 30 ± 1 51 ± 20 51 ± 4 GP2 26 ±9 46 ± 5  48 ± 1Pre-treatment with Tz at 50 μg/ml for 24 h increased specific CTX byHer577-stimulated CTL versus untreated MCF-7, SKOV-3 and LNCaP by 16%,47% and 83%, respectively (P=0.19). A dose-dependent incrementalincrease in CTX against each tumor cell line was seen with 10 and 50μg/ml doses of Tz.

It can be concluded that Her577 is a newly described T cell-epitope fromHER2/neu identified by using a novel method for the discovery ofimmunogenic peptide(s) for vaccine development by assessing the bindingsite of Tz. There is a potential for combination immunotherapy withtherapeutic Ab and unique peptide vaccines derived from the Ab-bindingsite.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all patents and publications that are cited for any reason,including U.S. Provisional Application No. 60/714,865, on which priorityis based, are specifically and entirely incorporated by reference. Thespecification and examples should be considered exemplary only with thetrue scope and spirit of the invention embodied within the followingclaims.

The invention claimed is:
 1. A method for treating a patient havingtumor cells that express a Her2/neu antigen, the method comprising,administering to the patient in any order an immunotherapy consistingof: (a) a therapeutically effective amount of trastuzumab; and (b) atherapeutically effective amount of a peptide vaccine comprising aHer2/neu peptide antigen that consists of the amino acid sequence of SEQID NO: 17 (GP2=IISAVVGIL) or SEQ ID NO: 18 (GP2′=IVSAVVGIL), as the solepeptide antigen in the vaccine.
 2. The method of claim 1, wherein theHer2/neu peptide antigen is administered before, after or at the sametime as administration of the trastuzumab.
 3. The method of claim 1,wherein the patient is a breast cancer patient.
 4. The method of claim1, wherein the Her2/neu peptide antigen consists of the amino acidsequence of SEQ ID NO:
 17. 5. The method of claim 1, wherein theHer2/neu peptide antigen consists of the amino acid sequence of SEQ IDNO:
 18. 6. The method of claim 1, wherein the Her2/neu peptide antigenis administered after the trastuzumab.
 7. The method of claim 4, whereinthe patient is a breast cancer patient.
 8. The method of claim 5,wherein the patient is a breast cancer patient.
 9. The method of claim7, wherein the Her2/neu peptide antigen is administered after thetrastuzumab.
 10. The method of claim 8, wherein the Her2/neu peptideantigen is administered after the trastuzumab.